Inflammation and immune responses play a central role in most of the major diseases that impact human health, including cancer, cardiovascular disease, autoimmunity, neuroinflammatory processes, diabetes and other diseases. In addition, immunomodulatory therapies and adoptive cell therapies have become established as powerful therapeutic approaches in cancer. The ability monitor and distinguish immune cell subsets (e.g. T and B lymphocytes, macrophages, neutrophils, etc.) noninvasively in vivo will be invaluable to the development of effective treatments. Positron emission tomography (PET) provides highly sensitive, non- invasive and quantitative imaging, which can be combined with antibody-based probes to provide molecular imaging of tissues and cells in vivo based on cell surface phenotype. Furthermore, engineered antibody fragments (minibodies and cys-diabodies) have been developed with optimal kinetics enabling rapid in vivo targeting and clearance for imaging applications by immunoPET. In the proposed work, immunoPET will be developed to address the challenge of direct imaging of immune cell subsets. Engineered antibody fragments will be optimized for in vivo detection, imaging, and quantitation of T lymphocytes that express CD8, a marker that is a hallmark of cytotoxic T cells. Specific Aim 1 will focus on production of stable, optimized anti-CD8 minibodies and cys-diabodies derived from rat anti-mouse monoclonal antibodies. Specific Aim 2 will establish the optimal imaging agent, dose, and imaging protocol for visualization of the lymphoid organs in normal (wild-type) mice. Specific Aim 3 will focus on quantitation of T cells in normal and immune-stimulated mice to determine the parameters and limits of detection in mouse models. Once validated, immunoPET probes for CD8 T cells will be valuable tools to study immune processes in mouse models, providing a deeper understanding of immune responses in living organisms, and will open the door to broad methods for imaging and quantitating immune cell subsets in preclinical models and eventually humans.